LOCUS       sequence-419008-        7458 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive or doxycycline-inducible expression of 
            NUbo-E3(48)-mCherry-P2A-3xFLAG-Ubc7 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7458)
  AUTHORS   Renz C, Asimaki E, Meister C, Albanese V, Petriukov K, Krapoth NC, 
            Wegmann S, Wollscheid HP, Wong RP, Fulzele A, Chen JX, Leon S, 
            Ulrich HD
  TITLE     Ubiquiton-An inducible, linkage-specific polyubiquitylation tool.
  JOURNAL   Mol Cell. 2024 Jan 18;84(2):386-400.e11. doi: 
            10.1016/j.molcel.2023.11.016. Epub 2023 Dec 15.
  PUBMED    38103558
REFERENCE   2  (bases 1 to 7458)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2023.11.016"; journalName: "Mol Cell"; date: 
            "2024-01-18- 18"; volume: "84"; issue: "2"; pages: "386-400.e11"
COMMENT     Sequence Label: pcDNA5-FRT-TO-NUbo-E3(48)-mCherry-P2A-3xFLAG-Ubc7
FEATURES             Location/Qualifiers
     source          1..7458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1107..1133
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             1107..1133
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1983..2690
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2128..2146)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2396..2415
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2709..2765
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2766..2831
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    2838..2862
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3410..3427)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3416..3640
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3773..3792)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3924..3971
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3979..5001
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3979..5001
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5131..5252
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5168..5187)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5222..5241
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5301..5317)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5301..5317)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5314..5336)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5325..5341
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5349..5355,5356..5373,5374..5379))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5394..5415
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5532..5549)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5703..6291)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5783..5802)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6462..7253,7254..7322))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7085..7104
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7323..7427)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttatgcaga tcttcgtgaa gaccctgacc ggcaagaccg
     1021 ccaccctgga ggtggagagc agcgacacca tcgacaacgt gaagagcaag atccaggaca
     1081 aggagggcat ccccggcagc ggcgcctacc cctacgacgt gcccgactac gccggcaacg
     1141 gcatcctgtg gcacgagatg tggcacgagg gcctggagga ggccagccgc ctgtacttcg
     1201 gcgagcgcaa cgtgaagggc atgttcgagg tgctggagcc cctgcacgcc atgatggagc
     1261 gcggccccca gaccctgaag gagaccagct tcaaccaggc ctacggccgc gacctgatgg
     1321 aggcccagga gtggtgccgc aagtacatga agagcggcaa cgtgaaggac ctgctgcagg
     1381 cctgggacct gtactaccac gtgttccgcc gcatcagcaa gactagtgag ctgcagagca
     1441 accagcaccc aagcgcacag cgtctgagcg caaccggtgt gaacgcacac ggtcgtccac
     1501 agggtagcac ccagaacgct ctgcgccgta ccggccgtgt gaacggcggc cacccagtga
     1561 ccacccagat ggtggagacc gtgcagaacc tggcccccaa cctgcacccc gagcagatcc
     1621 gctacagcct ggagaacacc ggcagcgtgg aggagaccgt ggagcgctac ctgcgcggcg
     1681 acgagttcag cttcccacca ggtttcgagc caagccgcgc tccaatgggt gccaacgctg
     1741 ctgtggacaa caacgccgcc ggtggtggtg agttcaacga cccccgcaag aagaacatga
     1801 tctgcgccga gaacctgctg gacaagttcc acgtggacct gaacgaggac atgagcaacc
     1861 tgagcttcaa ggacctggac atcgaggagc gcaagcgcct gctggtgtgg caggcccgca
     1921 agaacctgga gaccaagctg cagagcgaca aggacctgca gagcctgctg accttaatta
     1981 acatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
     2041 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     2101 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
     2161 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
     2221 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
     2281 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg
     2341 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
     2401 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
     2461 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg
     2521 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg
     2581 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     2641 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaaa ttaattaacg
     2701 gaagcggagc cacgaacttc tctctgttaa agcaagcagg agacgtggaa gaaaaccccg
     2761 gtcctgacta caaagaccat gacggtgatt ataaagatca tgacatcgac tacaaggatg
     2821 acgatgacaa ggatatcaca agtttgtaca aaaaagcagg ctccaccatg agcaagaccg
     2881 cccagaagcg cctgctgaag gagctgcagc agctgatcaa ggacagcccc cccggcatcg
     2941 tggccggccc caagagcgag aacaacatct tcatctggga ctgcctgatc cagggccccc
     3001 ccgacacccc ctacgccgac ggcgtgttca acgccaagct ggagttcccc aaggactacc
     3061 ccctgagccc ccccaagctg accttcaccc ccagcatcct gcaccccaac atctacccca
     3121 acggcgaggt gtgcatcagc atcctgcaca gccccggcga cgaccccaac atgtacgagc
     3181 tggccgagga gcgctggagc cccgtgcaga gcgtggagaa gatcctgctg agcgtgatga
     3241 gcatgctgag cgagcccaac atcgagagcg gcgccaacat cgacgcctgc atcctgtggc
     3301 gcgacaaccg ccccgagttc gagcgccagg tgaagctgag catcctgaag agcctgggct
     3361 tctgagcggc cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg
     3421 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     3481 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     3541 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     3601 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     3661 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     3721 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     3781 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3841 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     3901 tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt
     3961 ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
     4021 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
     4081 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
     4141 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
     4201 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
     4261 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
     4321 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
     4381 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
     4441 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
     4501 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
     4561 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
     4621 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
     4681 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
     4741 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
     4801 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
     4861 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
     4921 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
     4981 cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat
     5041 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     5101 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     5161 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     5221 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     5281 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     5341 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     5401 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5461 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5521 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5581 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5641 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5701 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5761 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5821 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5881 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5941 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     6001 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6061 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     6121 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     6181 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6241 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6301 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6361 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6421 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6481 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6541 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6601 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6661 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6721 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6781 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6841 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6901 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6961 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     7021 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     7081 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     7141 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     7201 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     7261 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7321 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7381 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7441 aaagtgccac ctgacgtc
//
